Neuronal death in prion disorders is believed to result from a conformationally transformed, scrapie isoform (PrPSc) of the normal host prion protein (PrPC). The high correlation between PrPSc deposits and neurodegeneration has led to a cause and effect hypothesis. However, presence of neurodegeneration and transmission of prion diseases without detectable PrPSc suggest the presence of alternative mechanisms of neuronal death. Our long-term goal is to investigate potentially neurotoxic pathways of metabolism of normal and mutant PrP that initiate neurotoxicity without significant prpSC deposition. Recently, we have identified novel pathways of processing and turnover of mutant PrP with a stop codon at residue 145 (PrP'45), associated with a familial prion disorder. We believe that PrP'45 is neurotoxic through intracellular pathways. In particular, our data show that PrPi45 is degraded by the proteasomal pathway, and aggregates intracellularly. Surprisingly, a significant amount of PrP'45 is also rnistargeted to the nucleus. We hypothesize that cytotoxicity in this case is caused by perturbation of cellular metabolism by these unconventional pathways of PrP metabolism. Since fragments similar to PrP'45 are also generated by atypical processing of prpc and other mutant PrPs, the central goal of the present proposal is to analyze the cellular events leading to neurotoxicity by the abnormal accumulation of PrPt4s in the nucleus, and pathways of generation of similar PrP fragments in neuronal cells. In the first aim, we will identify specific nuclear localization signal(s) and the mechanism of transport of PrP'45 to the nucleus. The second aim will focus on whether accumulated PrP in the nucleus alters transcriptional activity that is physiologically relevant. In the third aim, we will determine if rnistargeted PrP is bound to specific nuclear proteins, and whether this association is biologically significant, and finally, we will analyze the mechanism(s) of generation of PrP fragments similar to PrPt45 from prpc or other mutant PrPs, since these would cause neurotoxicity in a manner similar to PrP'45. The experiments designed will use a variety of cell- and molecular biology techniques. In vitro nuclear transport, cell viability assays, in vitro translation, co-immunoprecipitation, Western blots, confocal immunomicroscopy and cell sorting will be used for the studies proposed in specific aims 1 and 2. For aims 3 and 4, Far Western analysis, electrophoretic mobility shift assay, in vitro transcriptional run-off assay, and differential mRNA display will be carried out. Our studies will provide a cell biological explanation for neurotoxicity of PrP that operates either concomitant with, or prior to PrPSc deposition, and help in developing strategies to disrupt these abnormal pathways of PrP metabolism.